EGFR inhibitors promote axon regeneration

ABSTRACT

Compositions and methods for promoting neural regeneration in a patient determined to have a lesion in a mature CNS neuron are disclosed. The method comprises the step of contacting the neuron with an EGFR inhibitor sufficient to promote regeneration of the neuron.

This work was supported by Federal Grant Nos. R21NS041999 and DA015335.The U.S. government may have rights in any patent issuing on thisapplication.

FIELD OF THE INVENTION

The field of the invention is the use of epidermal growth factorreceptor (EGFR) inhibitors for promoting neural regeneration of lesionedmature CNS neurons.

BACKGROUND OF THE INVENTION

Failure of successful axon regeneration in the CNS is attributed notonly to the intrinsic regenerative incompetence of mature neurons, butalso to the environment encountered by injured axons. The inhibitoryactivity is principally associated with components of CNS myelin andchondroitin sulfate proteoglycans (CSPGs) in the glial scar at thelesion site (1-4). Recent studies suggested that three myelin proteins,myelin-associated glycoprotein (MAG), Nogo-A and oligodendrocyte myelinglycoprotein (OMgp), collectively account for the majority of theinhibitory activity in CNS myelin (4-6). The inhibitory activity of MAG,OMgp and the extracellular domain of Nogo-A may be mediated by a commonreceptor complex that consists of the ligand-binding Nogo-66 receptor(NgR) and its signaling co-receptors p75/TROY and Lingo-1 (7-13).However, little is known about how signaling events occurring at theaxonal membrane are translated into specific cytoskeletal rearrangementsunderlying inhibition of axon regrowth. For instance, it is known thatMAG and perhaps other myelin inhibitors are able to induce an elevationof intracellular Ca²⁺ levels (14-16). But it is unclear howintracellular Ca²⁺ signaling may be involved in the inhibition of axonregeneration.

The involvement of EGFR activation in development and differentiation ofCNS neurons has been studied extensively. Goldshmit et al. (J Biol Chem(2004) 279:16349-16355) report that overexpression of SOCS2 in CNSneurons promotes neurite outgrowth, and that this outgrowth is blockedby addition of EGFR inhibitors PP3 and AG490. Wu et al. (Mol Biol Cell(2004) 15:2093-2104) report that the chondroitin sulfate proteoglycanversican V1 induces NGF-independent neuronal differentiation andpromotes neurite outgrowth in cultured PC12 cells by enhancing EGFR andintegrin activities, and that addition of the EGFR inhibitor AG1478significantly blocks differentiation. Wildering et al. (J Neurosci(2001) 21:9345-9354) report that EGF promotes axonal regeneration ofneurons of the crushed right internal parietal (RIP) nerve in the pondsnail Lymnaea stagnalis and that inhibition of EGF action by thespecific EGFR inhibitor PD153035 counteracts the effect of EGF on axonalregeneration. Li et al. (J Neurosci (2003) 23:6956-6964) report thatPC12 cell lines with reduced EGFR signaling have reduced neuriteoutgrowth in response to NGF and that AG1478, a specific EGFR tyrosinekinase inhibitor, is cytotoxic to these cells.

In light of these reports our finding that suppressing EGFR functionpromotes significant regeneration of a lesioned adult CNS neuron in thepresence of myelin inhibitory molecules was quite unexpected.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for promoting neuralregeneration of a lesioned CNS neuron. In one embodiment, the inventionprovides a method of promoting neural regeneration in a patientdetermined to have a lesion in a mature CNS neuron, the methodcomprising the step of contacting the neuron with an EGFR inhibitorsufficient to promote regeneration of the neuron.

In particular embodiments, the lesion is an axon lesion.

In particular embodiments, the lesion results from a traumatic injury,CNS degeneration, an optic nerve injury, or glaucoma.

In particular embodiments, the lesion results from a traumatic injury,and the contacting step is effected within 96, 48, or 24 hours offormation of the lesion.

In a particular embodiment, the lesion results from an acute spinal cordinjury, and the method optionally comprises contacting the neuron withmethylprednisolone sufficient to reduce inflammation of the spinal cord.

In a particular embodiment, the EGFR inhibitor is a small moleculeselected from the group consisting of erlotinib, gefitinib, GW2016,GW572016, PKI166, CL-1033, EKB-569, and GW2016.

In another embodiment, the EGFR inhibitor is a monoclonal antibodyselected from the group consisting of cetuximab, panitumumab, TheraCIM,EMD 72000, and MDX447.

In particular embodiments the EGFR inhibitor is administered to thepatient orally or by injection.

In another embodiment, the EGFR inhibitor is contained within animplantable device.

In a particular embodiment, the method further comprises the step ofdetecting a resultant neural regeneration, and optionally the neuralregeneration is detected inferentially by neurological examination.

A further aspect of the invention is the use of an EGFR inhibitor forthe manufacture of a medicament to promote neural regeneration in apatient determined to have a lesion in a mature CNS neuron.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The invention provides methods and compositions for promoting neuralregeneration of a lesioned CNS neuron. As used herein, the singularforms “a,” “an,” and “the,” refer to both the singular as well asplural, unless the context clearly indicates otherwise. For example, theterm “a CNS neuron” includes both single and multiple neurons and can beconsidered equivalent to the phrase “at least one CNS neuron.” Inpreferred embodiments, the neuron is a mammalian neuron, and inparticular embodiments is a human neuron.

The lesioned neuron is subject to growth inhibition by endogenous myelingrowth repulsion factors and may be in situ, i.e. located within thebrain, brainstem, spinal cord, or optic nerve of a patient or animalmodel, or in vitro co-cultured with oligodendrocyte myelin or withisolated myelin growth repulsion factors such as myelin-associatedglycoprotein (MAG), Nogo-A, and oligodendrocyte myelin glycoprotein(OMgp). The lesion may present at any part of the neuron. In particularembodiments a neurite (i.e. axon and/or dendrite) is lesioned, and theEGFR inhibitor treatment promotes neurite outgrowth. In one aspect, theinvention provides a method of promoting neural regeneration in apatient determined to have a lesion in a mature (i.e.terminally-differentiated, non-embryonic) CNS neuron, preferably apost-gestational, juvenile, pediatric or adult CNS neuron, the methodcomprising the step of contacting the neuron with an EGFR inhibitorsufficient to promote regeneration of the neuron. The patient is amammal such as a companion animal (dog, cat, etc.), livestock, animalmodel for neurodegeneration or CNS injury (e.g. rat, mouse, primate,etc), etc. In particular embodiments the patient is human.

The lesion can result from traumatic injury, stroke, pressure build-up,chronic neurodegeneration, etc. In a particular embodiment, the lesionresults from acute or traumatic injury such as caused by contusion,laceration, acute spinal cord injury, etc. In this embodiment, thecontacting step is preferably initiated within 96 hours of formation ofthe lesion, and more preferably within 72, 48, 24, or 12 hours. The EGFRinhibitor can be administered to the injured neuron in combination with,or prior or subsequent to, other treatment regimes such as the use ofanti-inflammatory agents. In a specific embodiment, the lesion resultsfrom acute spinal cord injury and the method additionally comprisescontacting the neuron with methylprednisolone sufficient to reduceinflammation of the spinal cord. In another embodiment, the lesionresults from neurodegeneration which, for example, can be caused byneurotoxicity or a neurological disease or disorder such as Huntington'sdisease, Parkinson's disease, Alzheimer's disease, multiple systematrophy (MSA), glaucoma, etc.

The EGFR inhibitor can be any inhibitor that specifically suppressesEGFR function, including antisense and RNAi oligonucleotide inhibitors,peptide nucleic acids, peptide antagonists, monoclonal antibodies (MAB),small molecule inhibitors (SMI), etc. In addition, various moleculesknown to interfere with EGFR signaling such as EGFR truncations, Gene 33polypeptide (also called RALT and MIG6), and kekkon can be targeted,introduced or expressed in target cells. For example, a wide variety oftechnologies are available for protein transfection, including the useof cationic liposomes, calcium phosphate coprecipitation,electroporation, microinjection, viral vectors, and a large number ofcommercially-available, proprietary lipid, polyamine and amphotericprotein reagents, including “ProJect”, “TransIT”, “Profect-1”, “Chariot”and ProteoJuice”. In a particular embodiment, the EGFR inhibitor is asmall molecule or monoclonal antibody that specifically suppresses thekinase function of EGFR. Table I lists several small molecule andantibody EGFR inhibitors that are FDA-approved or are in clinicaltrials.

TABLE I Drug Type Company Regulatory Status Cetuximab MAB ImcloneApproved Erlotinib SMI OSI-Pharmaceuticals Approved Gefitinib SMIAstraZeneca Approved Panitumumab MAB Abgenix Phase II TheraCIM MAB YMBiosciences Phase II EMD 72000 MAB Merck Phase I MDX447 MABMedarex/Merck Phase I GW572016 SMI GlaxoSmithKline Phase II PKI166 SMINovartis Phase II Cl-1033 SMI Pfizer Phase II EKB-569 SMI Wyeth Phase IGW2016 SMI GlaxoSmithKline Phase I

The EGFR inhibitor can be contacted with the neuron using any suitabledrug delivery method. For in vitro methods, the inhibitor is added tothe culture medium, usually at nanomolar or micromolar concentrations(see Examples 1 and 3). For in situ applications, the EGFR inhibitor canbe administered orally, by intravenous (i.v.) bolus, by i.v. infusion,intracranially, intraperitoneally, intraventricularly, by epidural, etc.Suitable protocols for administration of the EGFR inhibitor to a patientcan be readily derived from the extensive animal studies and clinicaltrials that have been conducted on EGFR inhibitors for the treatment ofcancer. In certain embodiments, the EGFR inhibitor is administeredorally or intravenously. Several orally- and intravenously-administeredEGFR inhibitors have shown to be well-tolerated and efficacious inglioma and other brain tumors, demonstrating that EGFR inhibitorsdelivered by these routes have a therapeutic effect on cells of the CNS.Small molecule EGFR inhibitors are typically administered orally atabout 50-500 mg/day, and monoclonal antibodies are typicallyadministered weekly by infusion at about 1-5 mg/kg body weight. In otherembodiments, the EGFR inhibitor is contained within an implantabledevice specifically adapted for delivery to a CNS neuron. The devicesinclude controlled release biodegradable matrices, fibers, pumps,stents, adsorbable gelatin (e.g. Gelfoam) or other devices loaded withpremeasured, discrete and contained amounts of an EGFR inhibitorsufficient to promote neuronal regeneration (see Example 5). In aparticular embodiment, the device provides continuous contact of theneuron with the EGFR inhibitor at nanomolar or micromolarconcentrations.

The subject methods may further comprise the step of detecting aresultant neural regeneration. For in vitro applications, neuralregeneration can be detected by any routinely used method such as aneurite outgrowth assay (see Example 1). For in situ applications,neural regeneration can be detected using imaging methodologies such asMRI. More commonly, neural regeneration will be detected inferentiallyby neurological examination showing improvement in the patient's neuralfunction. The detecting step may occur at any time point afterinitiation of EGFR inhibitor treatment, e.g. at least one day, one week,one month, three months, six months, etc. after initiation of treatment.In certain embodiments, the detecting step will comprise an initialneurological examination and a subsequent neurological examinationconducted at least one day, week, or month after the initial exam.Improved neurological function at the subsequent exam compared to theinitial exam indicates resultant neural regeneration. The specificdetection and/or examination methods used will usually be based on theprevailing standard of medical care for the particular type of neurallesion being evaluated (i.e. trauma, neurodegeneration, etc.).

The invention also provides EGFR inhibitor-eluting or EGFRinhibitor-impregnated CNS-implantable solid or semi-solid devices.Examples of CNS implantable devices include polymeric microspheres (e.g.see Benny et al., Clin Cancer Res. (2005) 11:768-76) or wafers (e.g. seeTan et al., J Pharm Sci. (2003) 4:773-89), biosynthetic implants used intissue regeneration after spinal cord injury (reviewed by Novikova etal., Curr Opin Neurol. (2003) 6:711-5), biodegradable matrices (see e.g.Dumens et al., Neuroscience (2004) 125:591-604), biodegradable fibers(see e.g. U.S. Pat. No. 6,596,296), osmotic pumps, stents, adsorbablegelatins (see e.g. Doudet et al., Exp Neurol. (2004) 189:361-8), etc.Preferred devices are particularly tailored, adapted, designed ordesignated for CNS implantation. The implantable device may contain oneor more additional agents used to promote or facilitate neuralregeneration. For example, in one embodiment, an implantable device usedfor treatment of acute spinal cord injury contains an EGFR inhibitor andmethylprednisolone or other anti-inflammatory agent. In anotherembodiment, the implantable device contains an EGFR inhibitor and anerve growth factor or hormone that promotes neural cell survival,growth, and/or differentiation, such as brain-derived neurotrophicfactor (BDNF), ciliary neurotrophic factor (CNTF), nerve growth factor(NGF), etc.

EXAMPLE 1 EGFR Inhibitors Promote Neurite Outgrowth on a MyelinSubstrate

We screened approximately 400 well-characterized small molecules in aneurite outgrowth assay of cerebellar granule cells (CGNs) onimmobilized myelin substrate. Myelin was immobilized on 96-well platesusing published methods (8). Individual compounds from a signalingmolecule targeted drug library (TOCRIS) were diluted by 10-folddilutions centered on their IC₅₀ values, in concentrations typicallyvarying from low nanomolar to micromolar, and transferred to culturewells with 5×10⁴ CGNs per well. Cells were grown overnight, fixed inparaformaldehyde, and stained with tubulin antibodies. Outgrowth wasindependently scored by two blind observers. Positively scored compoundswere picked for further verifications. Each compound was evaluated at a10⁴ dilution range centered on its IC₅₀ value to ensure effectiveconcentrations for each drug tested. Several internal controls,including cAMP analogues, phosphodiesterase inhibitors, and aRho-associated protein kinase inhibitor were identified in the screen,confirming previous findings and validating our approach. The majorityof compounds tested did not have a noticeable effect on neuriteoutgrowth, and a small number of them were toxic. Surprisingly, severalEGFR kinase inhibitors, including Tyrphostin B44(−), Tyrphostin A47 andTyrphostin A46, showed the greatest ability to counter the effects ofmyelin inhibition, suggesting that EGFR kinase activity might play animportant role in transducing myelin-dependent CNS outgrowth inhibitionsignals in neurons.

To confirm the involvement of EGFR kinase in myelin inhibition, wetested two well-characterized EGFR inhibitors with distinctivemechanisms of action, AG1478 and PD168393, and a non-receptor tyrosinekinase inhibitor, AG1288, in the outgrowth assay. P7-9 CGNs were platedon control and immobilized myelin substrate and grown in the presence ofAG1288 (1 μM), AG1478 (10 nM) and PD168393 (10 nM) for 20 hr (8, 10).Cells were fixed in paraformaldehyde and stained with anti-tubulinantibody (Tuj1, Covance) to visualize and quantitate neurite length. TheEGFR inhibitors AG1478 and PD168393, but not AG1288, effectivelypromoted neurite outgrowth from both CGNs and dorsal root ganglion (DRG)neurons when grown on substrates of whole myelin as well as individualmyelin inhibitors, including Nogo-66 and MAG. Approximately 500 neuronswere counted for each condition from at least 3 independent experiments.All AG1478 and PD168393 treatments on inhibitors were significant. Incontrast, none of the treatments affected either the neurite outgrowthon a control poly-D-lysine (PDL) substrate or neuronal survival.Similarly, EGFR kinase inhibitors were able to block myelin neuriteoutgrowth inhibition in retinal explant cultures grown within a collagenmatrix laden with myelin. P5-6 mouse retinas were dissected and cut intosmall explants on a tissue chopper. The resultant explants were culturedwithin a collagen matrix with and without myelin (10 μg/ml) in thepresence or absence of PD168393 (10 nM) for 72 hrs. Explants were fixedand stained with anti-tubulin antibodies. PD168393 added to myelincultures significantly promoted outgrowth of explants when compared tomyelin alone (*Student's t test P<0.0001). These results indicated thatEGFR inhibitors do not activate a general neurite outgrowth program, butrather interfere with signaling pathways required for the activity ofmyelin inhibitors.

EXAMPLE 2 CGNs Overexpressing Mutant EGFR and Grown on Myelin ExhibitExtensive Neurite Outgrowth

To complement our results obtained from pharmacological manipulations,we made recombinant herpes simplex viruses (HSVs) that transduceexpression of a mutant form of human EGFR in neurons (18, 19). Thekinase deficient EGFR-K721A (kdEGFR) carries a point mutation in thekinase ATP-binding site (18, 19), and when over-expressed, can inhibitthe activity of endogenous EGFR in a dominant-negative manner. Wild type(wtEGFR) and kinase-deficient EGFR (kdEGFR)HSV infected 2-2 cells werestimulated with 1 ng/ml EGF for 5 min and the lysates were immunoblottedwith an antibody against pTyr1173 EGFR (Santa Cruz). Blots were strippedand reprobed with anti-EGFR antibodies. CGNs were infected with HSVviruses and plated on control and myelin substrates. The average neuritelengths were obtained. CGNs infected with mutant, but not wild typeEGFR, exhibited extensive neurite outgrowth on myelin. These resultsindicated that EGFR kinase activity is required for myelin dependentneurite outgrowth inhibition.

EXAMPLE 3 Myelin Inhibitors Nogo-66 and OMgp Activate EGFR

Previous genetic studies have implicated EGFR in neuronal migration andaxonal projection during development (20, 21); however, the expressionand function of EGFR in the adult nervous system remains unclear. Usingin situ hybridization, we found that EGFR was expressed in most parts ofthe mature nervous system, including the cerebral cortex, cerebellum,most DRG neurons, and retinal ganglion cells (RGCs). Furthermore,anti-EGFR antibodies immunostained both cell bodies and neurites ofcultured DRG neurons and retinal explants.

To examine whether myelin inhibitors directly influence the activity ofEGFR in myelin-responsive neurons, we treated serum-starved CGNs withrecombinant soluble myelin inhibitors and assessed the EGFR receptoractivity. By blotting neuronal lysates with antibodies directed againstphosphorylated EGFR we found that both Nogo-66 and OMgp at 5 nMtriggered EGFR phosphorylation as early as 1 min post-stimulation. ThisEGFR activation appeared to be specific for the myelin inhibitors asneither a control alkaline phosphatase (AP) protein nor thechemorepellant Semaphorin3A (Sema3A) induced detectable levels of EGFRphosphorylation. Additional evidence for EGFR activation was obtained byobserving that Nogo-66-dependent ERK1/2 MAP kinase activation occurredin an EGFR kinase-dependent manner

Previously, we developed a truncated form of NgR that has the ability tobind to ligands but not its signaling co-receptors (8-10, 12). Whenover-expressed, the truncated receptor can compete with endogenous NgRfor coreceptor binding and thus block inhibitor-induced signalingpathways (8-10, 12). We found that expression of this truncated, but notfull-length, NgR also efficiently blocked EGFR phosphorylation triggeredby Nogo-66, suggesting that EGFR activation is NgRcomplex dependent.Next, we performed cell surface binding and co-immunoprecipitationexperiments, but failed to detect either direct binding of EGFR toinhibitor ligands, or a physical association with NgR or p75. Theseresults indicated that EGFR is not a receptor for myelin-derivedinhibitors, nor is it likely part of the canonical NgR complex. It isknown that in addition to activation by its cognate ligands, EGFRphosphorylation can also result from “trans-activation” by othersignaling pathways (22, 23). For example, angiotensin II acts throughthe angiotensin AT1 receptor, to promote growth of cardiomyocytes viatrans-activation of the EGFR and activation of MAP kinase (23). Failureto detect EGFR in the NgR receptor complex suggests a possibletrans-activation of EGFR by signaling downstream of the active NgRreceptor complex. In support of this, we found that the extent of EGFRphosphorylation triggered by optimal concentrations of myelin inhibitorswas comparable to that resulting from low concentrations (1-2 ng/ml) ofepidermal growth factor (EGF), reminiscent of what has been previouslyreported for EGFR trans-activation (24). Several molecules have beenimplicated in EGFR trans-activation, including Ca²⁺, PKC, nonreceptortyrosine kinases (Src and Pyk2), G-protein-coupled receptors, andmetalloproteases that generate EGF-like ligands (22-25). We examinedwhether any of these mechanism(s) are involved in the NgR-dependent EGFRtrans-activation by inhibiting specific signaling pathwayspharmacologically. Only the calcium chelators EGTA and BAPTA-AMsignificantly decreased EGFR phosphorylation subsequent to Nogo-66treatment. A PKC inhibitor Go6976, metalloprotease inhibitors TAPI andGM6001, a Src inhibitor PP2, and pertussis toxin, had no effect onNogo-66 elicited EGFR phosphorylation. In further support of the idea ofEGFR trans-activation, neither EGTA nor BAPTA-AM had any effect ondirect EGFR activation by EGF. Thus, our results indicate EGFRtransactivation is a critical downstream component of Ca2+ signaling inresponse to myelin inhibitors.

EXAMPLE 4 EGFR Inhibitors Neutralize the Neurite Outgrowth InhibitoryActivity of CSPGs

In addition to myelin inhibitors, chondroitin sulfate proteoglycans(CSPGs) in the glial scar represent a major hurdle for regeneratingaxons. Consistent with previous observations that CSPGs elevateintracellular Ca²⁺ levels in responding neurons (26), we found that EGFRinhibitors could also neutralize the neurite outgrowth inhibitoryactivity of CSPGs. Retinal explants were grown in collagen gels with andwithout CPSGs (200 ng/ml, Chemicon) and PD168393 (100 nM) for threedays, fixed and stained with anti-tubulin antibodies. PD168393significantly increased neurite length of explants when compared toCSPGs alone. Furthermore, soluble CSPG added to serum-starved CGNs wasable to elicit EGFR phosphorylation in a calcium-dependent manner. Incontrast, neither growth cone collapse nor repulsive responses inducedby Sema3A were affected by EGFR inhibitors. Since Sema3A has previouslybeen suggested to act independently of intracellular Ca²⁺ (27), theseresults further indicate that EGFR is a critical calcium-specificsignaling molecule in axon guidance pathways.

EXAMPLE 5 EGFR Inhibition Promotes Regeneration of Lesioned Optic NerveFibers in Adult Mice

The finding that inhibiting neuronal EGFR activity could efficientlyblock the inhibitory activity of both myelin inhibitors and CSPGsprompted us to examine whether EGFR inhibitors introduced at a CNSlesion site could promote axon regeneration in a model of optic nervecrushing (28). All animal experiments were done in accordance withprotocols approved by the institutional animal care and use committee atSchepens Eye Research Institute. Adult mouse optic nerves were exposedbehind the eyeball and crushed. Immediately after injury in adult mice,Gelfoam soaked in a solution containing the EGFR inhibitor PD168393 or0.1% DMSO (control) was placed against the crush site of the nerve andreplaced every three days for the first six days of the study. Animalswere sacrificed two weeks post injury followed by transcardial perfusionwith 4% paraformaldehyde. Optic nerves were cryosectioned at 10 μm andstained with an anti-GAP43 antibody (Chemicon) to detect regeneratingaxons (28). Little regeneration was detected in DMSO-treated controlmice. However, suppression of EGFR kinase activity by injury siteapplication of PD168393 resulted in substantial axonal regrowth with a9-fold increase in the number of regenerating axons, measured 0.25 mmbeyond the injury site, compared to control mice. To test thepossibility that the observed axon regrowth of retinal ganglion neurons(RGCs) after PD168393 treatment was a consequence of improved cellsurvival, we stained retinal sections with the anti-tubulin Tuj1antibody, which stains RGCs in the retina, and counted surviving RGCs.No detectable effect of PD168393 on RGC survival was found. Thus,blocking EGFR activity locally and within a short time window followinginjury is sufficient to promote significant regeneration of lesionedoptic nerve fibers in adult mice.

EXAMPLE 6 Neuroprotective Effect of Gefitinib after Cortical ImpactInjury in Rats

Using methodology adapted from Cherian et al. (J Pharmacol Exp Ther.(2003) 304:617-23), the effects of different doses and treatmentschedules of gefitinib on a rat model of brain impact injury are tested.A total of 60 male Evans rats weighing 300 to 400 g are assigned to oneof the following doses injected intraperitoneally (i.p.): none (salinecontrol group), 1, 10, and 50 mg/kg/day gefitinib. The rats are furtherassigned to a treatment duration of 1, 3, 7, or 14 days, with 4 rats ineach treatment group, and 3 rats in each control group (i.e. salineadministered for 1, 3, 7, or 14 days).

The details of the methods to produce the impact injury have beenpreviously described (Cherian et al., J. Neurotrauma (1996) 13:371-383).Briefly, the head of the rat is fixed in a stereotaxic frame by ear barsand incisor bar. A 10-mm diameter craniotomy is performed on the rightside of the skull over the parietal cortex. An impactor tip having adiameter of 8 mm is centered in the craniotomy site perpendicular to theexposed surface of the brain at an angle of approximately 45 degrees tothe vertical. The tip is lowered until it just touches the duralsurface. The impactor rod is then retracted, and the tip advanced anadditional 3 mm to produce a brain deformation of 3 mm during theimpact. Gas pressure applied to the impactor is adjusted to 150 psi,giving an impact velocity of approximately 5 m/s and duration ofapproximately 150 to 160 ms.

Rats are fasted overnight and anesthetized with 3.5% isoflurane in 100%oxygen in a vented anesthesia chamber. Following endotracheal intubationwith a 16-gauge Teflon catheter, the rats are mechanically ventilatedwith 2% isoflurane in 100% oxygen for the surgical preparation and forthe impact injury. Intracranial pressure (ICP) is monitored by a 3Fmicrosensor transducer (Codman & Schurtleff, Randolph, Mass.) insertedin the left frontal lobe, well away from the impact site. ICP ismonitored during the impact injury as a measure of the severity of theinjury. Rectal temperature is maintained at 36.5-37.5° C. by a heatingpad, which is controlled by rectal thermistor. Brain temperature is keptconstant at 37° C. with the help of a heating lamp directed at the head.

Each dose of gefitinib is dissolved in 1 ml of sterile 0.9% saline sothat the volume delivered is the same for each group and only the dosageof gefitinib varies. The first dose is administered within 1 hourfollowing impact injury. After removing all catheters and suturing thesurgical wounds, the rats are allowed to awaken from anesthesia. For thefirst 3 days post injury, the rats are treated with butorphanoltartrate, 0.05 mg of i.m. every 12 h (twice a day), for analgesia andenrofloxacin 2.27%, 0.1 ml of IM qd, to reduce the risk of postoperativeinfections. Gefitinib is administered once daily for the assignedtreatment duration.

The outcome measures are performed by investigators who are blinded tothe treatment group. At 2 weeks after the impact, the animals are deeplyanesthetized with a combination of ketamine/xylazine/acepromazine andperfused transcardially with 0.9% saline, followed by 10% phosphatebuffered formaldehyde. The entire brain is removed and fixed in 4%formalin. The fixed brains are examined grossly for the presence ofcontusion, hematoma, and herniation. The brains are photographed,sectioned at 2-mm intervals, and then embedded in paraffin. Hematoxylinand eosin (H&E) stained 9-μm thick sections are prepared for histologicexamination. Particular care is made to include the largestcross-sectional area of cortical injury on the cut surface of theembedded sections. The H&E-stained coronal sections are digitized usinga Polaroid Sprint Scanner (Polaroid Corporation, Waltham, Mass.)equipped with a PathScan Enabler (Meyer Instruments, Houston, Tex.). Theinjury volume is measured by determining the cross-sectional area ofinjury in each H&E-stained coronal image and multiplying by thethickness of the tissue between the slices. This slab volume techniqueis implemented on the image processing program Optimas 5.2 (OptimasCorporation, Seattle, Wash.). Neurons in the middle 1-mm segments of theCA1 and CA3 regions of the hippocampus are counted at a magnification of200×. Neurons are identified by nuclear and cytoplasmic morphology, andindividual cells are counted whether normal or damaged. Neurons withcytoplasmic shrinkage, basophilia, or eosinophilia or with loss ofnuclear detail are regarded as damaged. The regions measured are 1 mmlong and 1 mm wide (0.5 mm on either side of the long axis of thesegment). The total number of neurons and the number of neurons thatappear normal are expressed as neurons per squared millimeter.

Gefitinib treatment regimes that demonstrate favorable neuroprotectiveeffect are repeated in follow-up studies on rats divided into groupsthat receive a first dose of gefitinib at 1, 24, 48, 72, or 96 hoursafter brain impact injury, and the time window for the neuroprotectiveeffect of gefitinib administration following traumatic brain injury isassessed.

EXAMPLE 7 Improved Neurological Outcome Following Cetuximab Treatmentfor Acute Spinal Cord Injury

We adapted our protocol for this study from the Sygen® Multicenter AcuteSpinal Cord Injury Study described by Geisler et al (Spine (2001)26:587-598). It is a prospective, double-blind, randomized, stratified,multicenter trial, randomizing approximately 800 patients so as to haveat least 720 completed and evaluable in each of three initial treatmentgroups: placebo, low-dose cetuximab, and high-dose cetuximab. Thepatients are stratified into six groups, according to three degrees ofinjury severity (American Spinal Injury Association grades A, B, andC+D) and two levels of anatomic injury (cervical and thoracic). Thetrial is sequential with preplanned interim analyses as each group of720/4=180 patients reach their 26-week examination and become evaluable.Patients are required to have at least one lower extremity with asubstantial motor deficit. Patients with spinal cord transection orpenetration are excluded, as are patients with a significant caudaequina, brachial or lumbosacral plexus, or peripheral nerve injury.Gunshot injuries that do not penetrate the cord are allowed. Multipletrauma is allowed as long as it is not so severe as to preventneurologic measurement evaluation or interpretation.

All patients are to receive the second National Acute Spinal Cord InjuryStudies (NASCIS II) dose regimen of methylprednisolone (MPSS) startingwithin 8 hours after the spinal cord injury (SCI). To avoid any possibleuntoward interaction between MPSS and cetuximab the study medication isnot started until after completion of MPSS administration.

The placebo group has a loading dose of placebo and then 56 days ofplacebo. The low-dose cetuximab group has a 300-mg loading doseadministered intravenously (i.v.) followed by 100 mg/day i.v. for 56days. The high dose cetuximab group has a 600-mg loading dose followedby 200 mg/day for 56 days.

The baseline neurologic assessment includes both the AIS and detailedAmerican Spinal Injury Association (ASIA) motor and sensoryexaminations. Modified Benzel Classification and the ASIA motor andsensory examinations are performed at 4, 8, 16, 26, and 52 weeks afterinjury. The Modified Benzel Classification is used for post-baselinemeasurement because it rates walking ability and, in effect, subdividesthe broad D category of the AIS. Because most patients have an unstablespinal fracture at baseline, it is not possible to assess walkingability at that time; hence the use of different baseline and follow-upscales. Marked recovery is defined as at least a two-grade equivalentimprovement in the Modified Benzel Classification from the baseline AIS.The primary efficacy assessment is the proportion of patients withmarked recovery at week 26. The secondary efficacy assessments includethe time course of marked recovery and other established measures ofspinal cord function (the ASIA motor and sensory scores, relative andabsolute sensory levels of impairment, and assessments of bladder andbowel function).

EXAMPLE 8 Gefitinib Reduces Neurodegeneration in Mouse Model of MultipleSystem Atrophy

Multiple system atrophy (MSA) is a neurodegenerative disease thataffects oligodendrocytes and CNS neurons. This study utilizes a recentlydeveloped mouse model of MSA (Yazawa et al, Neuron (2005) 45:847-859) toassess neurological outcome following gefitinib treatment. This MSAmodel is a transgenic mouse that overexpresses human α-synuclein, whichaccumulates in normal and degenerating axons and axon terminals inassociation with oligodendroglia and neuron loss and slowly progressivemotor impairments. Mice are assigned to one of the following dosesinjected intraperitoneally (i.p.): none (saline control group), 1, 10,and 50 mg/kg/day gefitinib. Treatment is initiated with 1 month oldmice. At 3, 6, 12, 18, and 24 months of age, motor testing is performedon gefitinib-treated and control mice using the rotarod treadmill testand wire hanging grip strength test. Mice are sacrificed at 12 and 24months of age, brain sections are fixed, neural cell morphology isanalyzed by transmission electron microscopy (EM), and the total numberof neurons is counted.

Rotarod Treadmill Test: The accelerating rotarod treadmill (Ugo Basile,Italy) is used to analyze motor function of treated and control mice.Mice are given three trials with 45 min intertrial intervals on each of2 consecutive days for 3 weeks. Each animal's endurance time (AET) isrecorded, and the average of AETs is calculated.

Wire Hanging Grip Strength Test: Mice are placed with their forepaws ona horizontal wire and allowed to grasp the wire and remain suspended.Each mouse is given two trials with an intertrial interval of 2 hr. Thetotal time the mice remain hanging on the wire is recorded as hangingtimes in seconds.

Transmission EM: Mice at 12 and 24 months of age (n=3 of each) areanalyzed by transmission EM. The mice are deeply anesthetized andsacrificed by cardiac perfusion using 0.1 M cacodylate buffer (pH 7.4),followed by 4% paraformaldehyde and 2% glutaraldehyde. Cerebrum, pons,cerebellum, and lower thoracic spinal cord are fixed for 18 hr. Tissuesare postfixed with 2% osmium tetraoxide for 1 hr and dehydrated andembedded in Epon. Ultra thin sections are cut and observed with a Joel1010 transmission electron microscope (Peabody, Mass.).

Quantitative Analysis of Neurons and Oligodendrocytes: The total numberof neurons in the L2 lumbar spinal cord as well as dopaminergic neuronsin the substantia nigra is counted on sections from 12 and 24-month-oldgefitinib-treated and control mice (each for n=3, 8 sections from eachmouse). Cells are visualized by immunostaining with NeuN (Chemicon,Temecula, Calif.), an antibody that labels neuronal nuclei, and tyrosinehydroxylase (TH) (Pel-Freese, Brown Deer, Wis.). Quantitative analysesare conducted on photographs covering the entire area of interest withimage analysis software, ImageProPlus (Media Cybernetics, Silver Spring,Md.). For oligodendrocyte analysis, cells are visualized byimmunostaining with anti-CNP antibody. The total number of cells in thelumbar spinal cord sections from the treated and control mice (each forn=3, 8 sections) is manually counted.

EXAMPLE 9 Neuroprotective Effect of EGFR-specific shRNA

A rat model for transient global ischemia demonstrates theneuroprotective effect of EGFR-specific short hairpin RNA (shRNA) usingmethodology adapted from Ning et al., J. Neurosci. (2004) 24:4052-60.

An EGFR target sequence corresponding to nucleotides 2529-2557 of EGFRmRNA (accession no. X00588; see Zhang et al., Clin Cancer Res. (2004)10:3667-77) is constructed into a pSUPER vector (see Brummelkamp et al,Science (2002) 296:550-553). A scrambled EGFR target sequence is used toconstruct control vectors. Plasmid transfections are done usingLipofectAMINE 2000 (Invitrogen, Burlington, Ontario, Canada) asdescribed previously (Wan et al., Nature (1997) 388:686-690).

Adult male Wistar rats weighing 200-300 gm are anesthetized with 2%halothane and placed in a David Kopf Instruments (Tujunga, Calif.)stereotaxic apparatus. EGFR-specific shRNA and vehicle are injected intothe CA1 pyramidal cell layer of rats at the following coordinates: 3.5mm posterior to bregma, 1.6 mm lateral from the midline, and at a depthof 3.0 mm from the skull surface. A volume of 1 μl of concentratedvector (1×10⁶ particles/μl) is injected into one side of the hippocampusat a rate of 0.05 μl/min.

Two days after plasmid injection the rats are anesthetized with 1.5%halothane, and the vertebral arteries are electrocauterized on day one.Twenty-four hours later, the animals are reanesthetized, and both commoncarotid arteries are clamped with aneurysm clips for 15 min. Animals areincluded for subsequent experiments only if they display an isoelectricEEG during the entire occlusion period, displayed a dilated pupil and alack of corneal light reflex, and recover EEG activity within 30 min ofreperfusion. Body temperature, as measured with a rectal thermometer, ismaintained between 36.5 and 38° C. throughout the procedure with aheating lamp. Sham animals receive identical surgical exposure andhandling without vessel occlusion.

At 48 hr after global ischemia, rats are perfused with 4%paraformaldehyde. Coronal sections (40 μm thick) through the dorsalhippocampus are cut on a sliding microtome and then processed forFluoro-Jade B histochemical staining, which consistently reveals dyingor degenerating neurons. Quantitative assessment is conducted byvisually counting Fluoro-Jade B-stained neurons using a 20× objectivelens. Average cell numbers are obtained by averaging counts over oneconsistent field at the middle point of the dorsal hippocampus wherevectors and vehicle are injected and over three sections (80 μm apart)in each brain. The neuroprotective effect of EGFR-specific shRNA isdemonstrated by a decrease in Fluoro-Jade B-stained neurons comparedwith controls.

The foregoing examples and detailed description are offered by way ofillustration and not by way of limitation. All publications and patentapplications cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims

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1. A method of promoting axonal regeneration in vitro by contactingmature CNS neurons that are cerebellar granule cells (CGNs), dorsal rootganglion cells (DRGs) or retinal ganglion cells (RGCs) following lesioninjury, with a small molecule epidermal growth factor receptor (EGFR)inhibitor sufficient to promote axonal regeneration.
 2. The method ofclaim 1 wherein the contacting is effected within 24 hours of formationof the injury.
 3. The method of claim 1 further comprising the step ofdetecting a resultant regeneration.
 4. The method of claim 1 wherein theEGFR inhibitor is selected from the group consisting of erlotinib,gefitinib, GW2016, GW572016, PKI166, CL-1033, EKB-569, and GW2016. 5.The method of claim 1 wherein the EGFR inhibitor is erlotinib.
 6. Amethod of promoting axonal regeneration in vivo in a mouse by contactingmature CNS neurons that are retinal ganglion cells (RGCs) followingoptic nerve crush injury in said mouse, with a small molecule EGFRinhibitor sufficient to promote axonal regeneration.
 7. The method ofclaim 6 wherein the contacting is effected within 24 hours of formationof the injury.
 8. The method of claim 6 further comprising the step ofdetecting a resultant regeneration.
 9. The method of claim 6 wherein theEGFR inhibitor is selected from the group consisting of erlotinib,gefitinib, GW2016, GW572016, PKI166, CL-1033, EKB-569, and GW2016. 10.The method of claim 6 wherein the EGFR inhibitor is erlotinib.
 11. Amethod of promoting axonal regeneration in vivo in a human by contactingmature CNS neurons that are retinal ganglion cells (RGCs) followingoptic nerve crush injury in said mouse, with a small molecule EGFRinhibitor sufficient to promote axonal regeneration.
 12. The method ofclaim 11 wherein the contacting is effected within 24 hours of formationof the injury.
 13. The method of claim 11 further comprising the step ofdetecting a resultant regeneration.
 14. The method of claim 11 whereinthe EGFR inhibitor is selected from the group consisting of erlotinib,gefitinib, GW2016, GW572016, PKI166, CL-1033, EKB-569, and GW2016. 15.The method of claim 11 wherein the EGFR inhibitor is erlotinib.
 16. Themethod of claim 11 wherein the contacting is effected within 24 hours offormation of the injury, the EGFR inhibitor is selected from the groupconsisting of erlotinib, gefitinib, GW2016, GW572016, PKI166, CL-1033,EKB-569, and GW2016, and the method further comprises the step ofdetecting a resultant regeneration.